Studies in vitro of the effects of adenosine 3′:5′-cyclic monophosphate on the phosphorylation of nuclear proteins in isolated rat heart nuclei

Abstract

Isolated rat heart nuclei were prepared by homogenization and sucrose-density-gradient centrifugation. The protein/DNA ratio of these nuclei was 3.1:1 (w/w), and the histones/non-histone proteins/DNA proportions were 1.4:1.6:1 (by wt.). Non-histone proteins were fractionated into six major groups by elution on a quaternized anion-exchanger (QAE-Sephadex A-50 column with increasing concentrations of NaCl in 5M-urea/0.01 M-Tris/HCl buffer (pH8.3). When isolated nuclei were incubated in a medium containing [gamma-32P]ATP, a differential distribution of 32P was observed in the six fractions of nonhistone proteins. The fractions eluted from the Sephadex column with 0.35M- and 0.6M-NaCl contained contained 80% of the total radioactivity incorporated into the non-histone proteins. This incorporation into the 0.35M- and 0.6M-NaCl fractions was increased by 66 and 112% respectively in the presence of cyclic AMP. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of these two particular fractions showed a selective increase in labelling of five protein bands in the presence of cyclic AMP.

Biphasic increase in the in vivo phosphorylation of nuclear 110-kDa protein during early lymphocyte transformation

“…Detection of higher-molecular-mass phosphoproteins has also been limited by the unsatisfactory separation methods often used. In recent years changes in the phosphorylation of high-molecular-mass polypeptides have been reported by different groups, for example in lung cells after toxic damage [Ill, in tumors as compared with normal tissues [12], in heart cells after in vitro CAMP incubation [13] or density-dependent in certain cell lines [14]. Correlation between phosphorylation of highmolecular-mass polypeptides and increase in cell activity is more evident in experiments investigating in vitro or in vivo phosphorylation of lymphocytes after short-term incubation with mitogens [15].…”

Myocardial chromatin activation in experimental hyperthyroidism in rats. Role of nuclear non-histone proteins.

“…The existence in rat myocardial nuclei of multiple, cyclic AMP-dependent protein kinases has also been reported by Akhtar and Itzhaki. 15 In view of the presence in nuclei of a non-histone protein phosphatase, 28 a system is available for the control of gene transcription by reversible phosphorylation of non-histone proteins.…”